Vol. 38: 259-266, 1987
1
MARINE ECOLOGY - PROGRESS SERIES
Mar. Ecol. Prog. Ser.
l
Published July 13
Fucoxanthin pigment markers of marine
phytoplankton analysed by HPLC and HPTLC
Simon W. wrightl & S. W. ~ e f f r e y ~
L
Antarctic Division, Department of Science, Channel Highway, Kingston, Tasmania 7150. Australia
CSIRO Division of Fisheries Research, Marine Laboratories, GPO Box 1538, Hobart, Tasmania 7001, Australia
ABSTRACT: High-performance thin-layer chromatography (HPTLC) and high-performance liquid
chromatography (HPLC) were used to separate fucoxanthin, 19'-hexanoyloxyfucoxanthin and a 19'butanoyloxyfucoxanthin-like pigment from algal cultures. Pavlova luthen (Prymnesiophyceae) contained fucoxanthin only, whereas Emiliania huxleyi, Phaeocystis pouchetii (Prymnesiophyceae) and
Pelagococcus subviridis (Chrysophyceae) contained all 3 fucoxanthin pigments in widely different
proportions. The 3 pigments, which were also found in phytoplankton samples from Antarctic waters,
may provide useful markers of phytoplankton in field samples. The HPTLC method provides a rapid
screening technique for fucoxanthin pigments, complementing the more powerful but time-consuming
HPLC
INTRODUCTION
Knowledge of the range of photosynthetic pigments
in a phytoplankton sample can indicate the algal
classes present in phytoplankton populations, information not available from simple chlorophyll and
'pheopigment' analyses (Jeffrey 1974). For example,
thin-layer chromatography
(TLC) unexpectedly
revealed significant quantities of chlorophyll b (green
algae) in the North Pacific Ocean (Jeffrey 1976), and
fucoxanthin (diatoms, prymnesiophytes) was found to
be the dominant carotenoid compared to the insignificant peridinin (photosynthetic dinoflagellates) in
Australian coastal and oceanic phytoplankton (Jeffrey
& Hallegraeff 1980, Hallegraeff 1981, Hallegraeff &
Jeffrey 1984, Jeffrey & Hallegraeff 1987). TLC (Jeffrey
1981) has also allowed separation and identification of
many types of chlorophyll degradation products in phytoplankton field samples (Hallegraeff & Jeffrey 1985).
The application of high-performance liquid chromatography (HPLC) in phytoplankton studies (e.g. Mantoura & Llewellyn 1983, Wright & Shearer 1984) has
extended the range of pigment separations, making
available more 'marker' pigments for finger-printing
algal types in the water column. Recent additions
include zeaxanthin, ascribed as a proposed marker for
cyanobacteria (Guillard et al. 1985), alloxanthin, a
marker for cryptomonads (Gieskes & Kraay 1983), and
prasinoxanthin, a marker for particular prasinophytes
(Foss et al. 1984).
Q Inter-Research/Printed in F. R. Germany
Fucoxanthin (Fig. 1) is confined in the phytoplankton
to diatoms, prymnesiophytes, raphidophytes and chrysophytes (Jeffrey & Vesk 1981, Stauber & Jeffrey
unpubl.). 19'-hexanoyloxyfucoxanthin is the major
carotenoid
of
the
coccolithophorid
Emiliania
(Coccolithus) huxleyi (Arpin et al. 1976) and was also
ascribed to natural blooms of the colonial prymnesiophyte Crymbellus aureus (Gieskes & Kraay,
1986a). Several dinoflagellates (e.g. Gyrodinium
aureolum; Bjerrnland & Tangen 1979, Tangen & B j ~ r n land 1981) which contain fucoxanthin and 19'-hexanoyloxyfucoxanthin may contain prymnesiophycean
endosymbionts (Bjerrnland et al. 1984). Recently, a new
fucoxanthin derivative from a Norwegian isolate of
Pelagococcus (Coc. min., Chrysophyceae) was unequivocally characterized as 19'-butanoyloxyfucoxanthin (Bjrarnland et al. 1985). The full paper on this
pigment has yet to be published. A similar pigment was
fucoxanthin
Fig. 1. Chemical structures of fucoxanthin, 19'-butanoyloxyfucoxanthin and 19'-hexanoyloxyfucoxanthin
260
Mar. Ecol. Prog. Ser. 38: 259-266, 1987
reported from other strains of Pelagococcus subviridis
(Lewin et al. 1977, Vesk & Jeffrey 1987), and is referred
to here a s a 19'-butanoyloxyfucoxanthin-like pigment.
Sucrose TLC was originally used to separate fucoxanthin and the 19'-butanoyloxyfucoxanthin-like pigment from Pelagococcus subviridis (Lewin et al. 1977).
In the present work, cellulose TLC (Jeffrey 1981) was
used to separate mixtures of fucoxanthin (from Pavlova
luthen]
and
19'-hexanoyloxyfucoxanthin (from
Emiliania huxleyi]. However, neither TLC system
resolved the 19'-hexanoyloxyfucoxanthin from the 19'butanoyloxyfucoxanthin-like pigment. Therefore a n
HPTLC (high-performance thin-layer chromatography)
method (Vesk & Jeffrey 1987) and the HPLC technique
of Wright & Shearer (1984) were both used to separate
all 3 fucoxanthin pigments. The present work reports
the use of both methods in cultured microalgae and
phytoplankton field samples from Antarctic waters.
MATERIALS AND METHODS
Algal cultures. The cultures used were from the
CSIRO Algal Culture Collection (Jeffrey 1980). Pavlova
lutheri (CSIRO Culture Code CS-23) and Emiliania
huxleyi (CS-57; Clone BT6 from Dr R. R. L. Guillard)
were used a s source material for standard fucoxanthin
(Berger et al. 1977) and standard 19'-hexanoyloxyfucoxanthin (Arpin et al. 1976) respectively. Pelagococcus subw.ridis (East Australian Current strain; CS99) was used as the source of the 19'-butanoyloxyfucoxanthin-like pigment (Vesk & Jeffrey 1987). These
cultures were grown in 125 m1 Erlenmeyer flasks at
17.5 "C in 40 ,uE m-' S-' white fluorescent light (Phillips
Daylight tubes) on 12: 12 h light: dark cycles in medium
f2 (Guillard & Ryther 1962). Light intensities were
measured with a Biospherical Optics light meter. Three
strains of Phaeocystis pouchetii (one isolated by J. L.
Stauber in 1981 from the East Australian Current [CS1651, one isolated by P. Thomas from Prydz Bay,
Antarctica in 1985. and one isolated by S. I. Blackbum
from Antarctic waters) were grown in G5 medium
(Loeblich & Smith 1968) under the same conditions.
Both strains on Antarctic P. pouchetii were grown at
5 "C. For larger volumes, 1 to l 0 l cultures were either
aerated, or shaken on an orbital shaker at 100 cycles
min-l, in Erlenmeyer flasks. None of the cultures used
in this study were completely axenic.
Collection of phytoplankton field samples. Samples
were collected from the MV Nella Dan every 1" latitude
from 60"s to the Antarctic coast, along 7 north-south
transects between 58" and 93"E longitude made during
January 1985 as part of the Second International BIOMASS Experiment, Phase 11 (SIBEX 11; Wright 1987). A
General Oceanics rosette sampler equipped with
twelve 5 1 Nishn bottles was used to collect water
samples from 0, 10, 25, 35, 50, 75, 100 and 200 m depth.
Between 2 and 4 1 of each sample were filtered for
pigment analysis.
Pigments: algal cultures. Cells were harvested at the
end of log phase by centrifuging either in a plankton
centrifuge at 8000 X g (large volumes) or in a bench
centrifuge at 2000 X g for 3 to 5 min (small volumes).
Pigments were extracted from the cell pellet in 100 %
acetone (except Pelagococcus subvin'dis, which
required freezing in ether/dry ice mixtures before
acetone extraction; Vesk & Jeffrey 1987).
For determination of chlorophylls a and c, spectrophotometric analyses of acetone extracts were
carried out using the appropriate equations of Jeffrey &
Humphrey (1975). Cell counts of the original cultures
were made using a haemocytometer.
For TLC, pigments were transferred from acetone to
peroxide-free diethyl ether by adding an equal volume
of ether to the acetone extract and gently mixing with
20 volumes of cold (5 "C) 5 % NaCl solution. The pigments migrated to the ether hyperphase, which was
collected, concentrated under a stream of NZ,dried
with a little solid NaC1, and stored at - 15 'C until use.
Pigments: phytoplankton field samples. Samples
were filtered under slight vacuum onto Whatman GF/F
filters (47 mm diameter). As magnesium carbonate can
bind pheophytins and chlorophyllides (Daley et al.
1973), it was not used as a filter aid. The filters were
immediately frozen in liquid nitrogen until ready for
analysis.
The frozen filters were cut into pieces of approximately 5 mm diameter, sonicated in 4 m1 methanol
(30 S at 50 W) using a Braun Labsonic 1510 sonicator
equipped with a 4 mm diameter needle probe and
filtered using the centrifugal system described in
Wright & Shearer (1984). Methanol was used as an
extraction solvent for HPLC because acetone caused
peak spreading. The filter debris was washed with
0.5 m1 methanol and recentrifuged. The combined
extract was filtered through a Millex-SR filter unit
(0.5 pm pore-size, Millipore Corp.), and chromatographed immediately. No significant allomerization of
chlorophylls occurred in the 10 min required for extraction.
High-performance
thin-layer chromatography.
Because the cellulose TLC plate (Jeffrey 1981) was
unable to separate all the fucoxanthin pigments, a 1dimensional reverse-phase HPTLC plate was used,
which successfully separated the 3 pigments. The system comprised a Merck RP-8 bonded silica plate,
developed for 30 min with methano1:water = 9:1 (v/v)
(Vesk & Jeffrey 1987).
High-performance liquid chromatography. Samples
Wright & Jeffrey: Fucoxanthin pigments of phytoplankton
of 250 p1 of extract were injected directly into a Waters
Associates liquid chromatograph. Two RCM-100 radial
compression modules containing Rad-Pak A cartridges
(5 pm particle size, octadecyl silicae) were used in
series, protected by a precolumn filter and an RCSS
Guard-Pak. The pigments were eluted using a linear
gradient from 90:10 acetonitri1e:water (v/v) to ethyl
acetate over 20 min at 2 m1 min-l. They were detected
by the sum of absorbances at 405 and 436 nm (Wright &
Shearer 1984), and integrated using a Waters Data
Module. For small peaks, where the signal-to-noise
ratio was low, the data module was found to be inaccurate, so these peaks were measured manually and
converted to pigment abundance using individual
calibration curves based on extinction coefficients of
160 l g-' cm-' (at 452 nm; solvent ethanol) for fucoxanthin and derivatives (Jensen 1966), 250 1
cm-' (at
450 nm; solvent ethanol) for carotene, diadinoxanthin
and diatoxanthin (Goodwin 1955), and 88.15 1g-' cm-'
(at 663 nm; solvent acetone for chlorophyll a (Jeffrey &
Humphrey 1975). As there was insufficient material in
most phytoplankton field extracts to obtain absorption
spectra, the pigments could be identified initially only
by comparison of retention times with known standards. However, after all field samples were analysed,
peak identities were confirmed by pooling selected
samples into one concentrate (referred to here as
Antarctic concentrate), which was rechromatographed.
Absorption spectra for initial identification were then
taken during elution, using a Hewlett-Packard 8450A
spectrophotometer.
Visible absorption spectra. For precise absorption
spectra, purified samples from cultures were isolated
on TLC systems (reverse-phase RP-8 plate, or washed
cellulose (Jeffrey 1981) and examined either in a Cary
Model 14 or a Shimadzu Model RP1 recording spectrophotometer.
It should be noted that where only chromatographic
and visible absorption spectroscopy data, and not full
chemical characterization, are available, the pigment
should be identified by the suffix '-like' (SCOR Working Group 77 on Photosynthetic Pigments, unpublished
recommendation). Thls has been followed in the present paper, e.g. only fucoxanthin from Pavlova luthen
and 19'-hexanoyloxyfucoxanthin from Emiliania huxleyi have been fully chemically characterized (Arpin et
al. 1976, Berger et al. 1977).
RESULTS
The reverse-phase RP-8 HPTLC plate separated the
3 fucoxanthin pigments - fucoxanthin (RF value 0.43),
19'-hexanoyloxyfucoxanthin (RF value 0.40) and the
19'-butanoyloxyfucoxanthin-like pigment (RF value
261
0.48) - from extracts of cultures of Pavlova l u t h e i
Emiliania huxleyi and Pelagococcus subviridis and
mixtures of these (Fig. 2A, B, C, D). All 3 pigments were
found in the East Australian Current strain and 2
Antarctic strains of Phaecystis pouchetii, although the
proportion of the fucoxanthin pigments was variable
(Fig. 2E, F, G). The Antarctic concentrate also contained the 3 fucoxanthin pigments (Fig. 2H).
0 -
0 0
A
B
Q
Q
C
D
0 0 0
E
F
G
@-""gin
H
0 Fucoxanthin d e r ~ v a t i v e s
0O t h e r c a r o t e n o i d s
Fig. 2. Separation of major carotenoids from various algal
species and the Antarctic concentrate on the reverse-phase
bonded-silica HPTLC plate (Merck.RP-8). (A) Pavlova lutheri;
(B) Emiliania hwdeyi; (C) Pelagococcus subviridis; (D) mixture
of A, B & C; (E, F, G) Phaeocystis pouchetii: East Australian
Current strain (EAC), and Antarctic strains (S.I. Blackburn
[Ant:SIB]and P. Thomas [Ant:PT]respectively);(H) Antarctic
concentrate. 1: peridinin (red); 2: 19'-butanoyloxyfucoxanthin-like (yellow-orange);3: fucoxanthin (red-orange);4: 19'hexanoyloxyfucoxanthin (yellow-orange);5: diadinoxanthn
(yellow);6: diatoxanthin (pale orange)
The visible absorption spectra of these pigments in
different solvents are given in Fig. 3 (no quantitative
relations between the solvents is intended) and absorption maxima and band ratios of the 3 fucoxanthin
pigments from different algal sources are given in
Table 1. The spectra of 19'-hexanoyloxyfucoxanthin
and the 19'-butanoyloxyfucoxanthin-like pigment
differed from that of fucoxanthin by having a pronounced minimum between Peaks I1 and I11 (sensu Ke
et al. 1970),whereas in fucoxanthin this minimum was
absent, Peak 111 being a shoulder. The 1II:II band ratios
for the 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin-like pigment differed depending on
algal source. We ascribe these variations to solvent
effects caused by slight differences in the concentration
Mar. Ecol. Prog. Ser. 38: 259-266, 1987
262
l
19'-he~an0ylo~yf~~0~anIhin-like-19'-butanoyloxyfucoxanthin-like
solvent:
diethyl ether
D
-
Fig. 3.Visible absorption spectra of fucoxanthin pigments in acetone, ethanol and &ethyl ether. (A) Fucoxanthin from Pavlova
lutheri; (B) 19'-hexanoyloxyfucoxanthin from Emiliania huxleyi; (C) 19'-butanoyloxyfucoxanthin-like pigment from
Pelagococcus subviridjs; (D) the 19'-hexanoyloxyfucoxanthn-like and 19'-butanoyloxyfucoxanthin-like pigments from
Phaeocystispouchetii in diethyl ether. (Band I1 is the 450 nm peak and Band I11 is the 470 nm peak referred to in Table 1; Ke et
al. 1970).
of water and other cell components extracted with the
pigments.
The separation of the 3 fucoxanthin pigments on the
HPLC system of Wright & Shearer (1984) is shown for
the first time in Fig. 4. The algal extracts and Antarctic
concentrate were the same as those used on the HPTLC
system (Fig. 2). HPLC confirmed the resolution of the
major fucoxanthin derivatives obtained with the
HPTLC plate, but the extra sensitivity also revealed the
presence of minor components, including the cis-isomers of fucoxanthin and 19'-hexanoyloxyfucoxanthin,
and an unknown carotenoid pigment, which eluted just
before fucoxanthin.
Quantitative analyses of the major pigments in Pavlova luthen, Emiliania huxleyi, Phaeocystis pouchetii
and the Antarctic samples are shown in Table 2, and
the ratios of the 3 fucoxanthin pigments are given in
Table 3.
Table 1. Absorption maxima (nm) and band ratios of fucoxanthin pigments in various solvents
Alga
CSIRO Culture
Code No.
Plgrnent
Solvent
Maxima
Band I1
Fucoxanthin
Ethanol
Acetone
Diethyl ether
448
446
446
Emiliania h uxleyi
19'-hexanoyloxyfucoxanthin
Ethanol
Acetone
Diethyl ether
447
445
444
Pelagococcus subvindis'
19'-butanoyloxyfucoxanthin-like. '
Ethanol
Acetone
Diethyl ether
446
445
444
Phaeocystispouchetli
19'-hexanoyloxyfucoxanthin-like' '
Ethanol
Acetone
Diethyl ether
446
444
444
19'-butanoyloxyfucoxanthin-like "
Ethanol
Acetone
Diethyl ether
447
445
444
Pavlova lutheri
''
CS-23
Band ratios
1II:II (O/O)
I
Band I11
-470
470
470
For a full analysis of P. subviridis pigments (2 strains),see Vesk & Jeffrey (1987)
Where only chromatographic and spectral data are available, and not full chemical characterization, the suffix-'like' is
added (SCOR Worlung Group 77, unpubl. recommendation)
0
3.3
Wnght & Jeffrey: Fucoxanthin pigments of phytoplankton
263
completely swamped the tiny amount of the 19'butanoyloxyfucoxanthin-like pigment available from
the field samples, for which no infrared spectra were
obtained at this time.
Other minor and trace pigments were also present in
the cultures and the Antarctic concentrate (Wright &
Shearer 1984, Vesk & Jeffrey 1987), but these will be
discussed fully elsewhere (Wright 1987).
DISCUSSION
Algal cultures
6
8
Pelagococcus
a
8
3
Phaeocystis (Ant.PT)
6
1
7
h
I
0
'
"
'
I
'
5
"
'
I
"
"
I
J
~
10
"
I
15
'
20
R e t e n t ~ o ntime ( m ~ n u t e s )
Fig. 4 . HPLC traces of pigment separations of the algae shown
In Fiq. 2 and the Antarchc concentrate Piqment fractions are.
1 19'-butanoyloxyfucoxanthin-like pigment; 2 fucoxanthn; 3
19'-hexanovlox~fucoxanthin;
4 cis-fucoxanthln; 5 a s 19'-he. .
xanoyloxyfucoxanthn; 6 diadinoxanthin: 7 dlatoxanthin; 8
chlorophyll a. Retention times a s shown. It should be noted
that Peak 3 in both Phaeocystis pouchetri isolated should b e
referred to a s '19'-hexanoyloxyfucoxanth~n-like',
untd chemical characterization has been carried out
That the pigments in the Antarctic concentrate were
identical with the fucoxanthin derivatives in the various algal cultures was confirmed by absorption spectroscopy and by CO-chromatographyin the HPLC and
HPTLC systems. In addition, allene groups, which are
present in fucoxanthin and its derivatives, were
detected in the fucoxanthin and 19'-hexanoyloxyfucoxanthin fractions isolated from the Antarctic samples
by a n absorption peak at 1929 nm, using infrared spectroscopy. Colourless contaminants (presumably lipids)
prevented full characterization of these pigments and
The present work confirms the presence of fucoxant h n alone (free from traces of other fucoxanthin pigments) in Pavlova lutheri, previously examined by
Berger et al. (1977). Emiliania huxleyi, in adhtion to
containing the dominant 19'-hexanoyloxyfucoxanthin
(Arpin et al. 1976), also contained small amounts of
fucoxanthn and the 19'-butanoyloxyfucoxanthin-like
pigment (Tables 2 & 3). Pelagococcus subviridis contained fucoxanthin and the 19'-butanoyloxyfucoxanthin-like pigment as the major carotenoids (Vesk &
Jeffrey 1987), but HPLC also revealed the presence of
small amounts of 19'-hexanoyloxyfucoxanthin (Fig. 2).
The identity of the fucoxanthin and 19'-hexanoyloxyfucoxanthln was confirmed by using pigment standards from well-characterized sources, but the identity
of the 19'-butanoyloxyfucoxanthin-like pigment from
Pelagococcus subviridis is stdl unclear. True 19'butanoyloxyfucoxanthn was chemically identified
together with fucoxanthin in the Norwegian isolate of a
Pelagococcus-like strain (Coc. min.) by Bj~lrnlandet al.
(1984),although the full data, including visible spectra
and chromatographic systems, have yet to be published. The fucoxanthin derivative present in both our
strains of Pelagococcus (Vesk & Jeffrey 1987) was
chromatographically different from 19'-hexanoyloxyfucoxanthn, yet spectrally similar (Fig. 3), a n d w e initially assumed it to be the 19'-butanoyloxyfucoxanthin
of Bj~lrnlandet al. (1984). However, in both our HPLC
and HPTLC systems, where the chromatographic
mobdity of an homologous series is related to the
length of the hydrophobic substituent, the sequence of
the 3 fucoxanthin pigments was different from that w e
would expect if this pigment were 'true' 19'-butanoyloxyfucoxanthin. O n the basis of chemical structures
(Fig. l ) , 19'-butanoyloxyfucoxanthin should b e intermediate in polarity between fucoxanthn and 19'-hexanoyloxyfucoxanthin, but in both our chromatographic systems (HPLC and HPTLC) it appeared to be
more polar than both (Fig. 2 & 3). The possibility
remains that our pigment either has a n addtional polar
group (e.g. hydroxyl) or has lost the methyl ester of the
264
Mar. Ecol. Prog. Ser. 38: 259-266, 1987
Table 2. Quantitative analyses of major pigments in Pavlova lutheri, Emiliania huxleyi, Phaeocystis pouchetii, Pelagococcus
s u b ~ d i and
s
the Antarctic concentrate, from HPLC analysis for carotenoids and spectrophotometric analysis (Jeffrey &
Humphrey 1975) for chlorophylls a and c. Minor carotenoids (Fig. 4) are not included
Pigment
P. lutheri
pg (10' cells)-'
Chlorophyll a
Chlorophyll c
p, p-carotene
Fucoxanthin
19'-hexanoyloxyfucoxanthin
19'-butanoyloxyfucoxanthin-like
Diadinoxanthm
Diatoxanthin
1
E. huxleyi
pg (10' cells)-'
Algal sample
P, pouchetii
P. subviridis
pg (10' cells)-' ~ i (10'
g cells)-'
Antarctic
concentrate &gI-'
31.0
8.0
0.39
0.41
31.3
0.27
4.3
2.4
Table 3. Ratios of fucoxanthin pigments from HPLC analysis of algal cultures and Antarctic water samples
I Alga or phytoplankton sample
Fucoxanthm
Pigment ratios
19'-hexanoyloxy19'-butanoyloxyfucoxanthin
fucoxanthin-like pigment
Pavlova lutheri
Emiliania huxleyi
Phaeocystis pouchetii (EA Current)
Phaeocystis pouchetii (Antarctic) '
Phaeocystis pouchetii (Antarctic)' '
Phaeocystis pouchetii (Antarctic)'
(senescent culture)
Antarctic concentrate
Average of 40 Antarctic water samples
I
''
P. Thomas isolate
S. Blackburn isolate
native fucoxanthin. On the basis of chromatographic
properties, this pigment is probably identical to the
unknown fucoxanthin derivative found by Gieskes &
Kraay (1986b) in the tropical Atlantic. The chemical
structure of this 19'-butanoyloxyfucoxanthin-like pigment from our strains of Pelagococcus subviridis is
currently under investigation.
Cultured strains of Phaeocystis pouchetii examined
in the present work contained all 3 fucoxanthin pigments, with the pigment chromatographically and
spectrally identical to 19'-hexanoyloxyfucoxanthin
dominant in each strain. The 19'-butanoyloxyfucoxanthin-like pigment was CO-dominantin the East Australian Current strain, but was present as a minor component with fucoxanthin in both Antarctic strains. Significant differences in the proportions of these pigments
were observed between strains and age of culture
(Table 3). These results are in agreement with those of
Dr S. Laaen-Jensen, who found both 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin in
cultured Phaeocystis pouchetii (pers. comm.). Our
results are in apparent contradiction to those of Gieskes
& Kraay (1986a), who stated that field populations of
Phaeocystis pouchetii do not contain 19'-hexanoyloxyfucoxanthin, but without further details it cannot be
determined whether culture, extractions methods or
strain differences would explain the conflicting observations.
Antarctic water samples
Extensive use of the HPLC method during the SIBEX
I1 experiment in Antarctic waters allowed distribution
of the 3 fucoxanthin pigments (integrated for the top
100 m) to be determined over a wide area (Wright
1987). Significant differences in the abundances of the
3 pigments were noted. The range of values for fucoxanthin was 2.2 to 247 mg m-2, compared to 5.1 to
323 mg m-2 for chlorophyll a, 2.4 to 83 mg m-2 for 19'-
I
Wright & Jeffrey: Fucoxanthin pigments of phytoplankton
hexanoyloxyfucoxanthin and 0.06 to 3.2 for the 19'butanoyloxyfucoxanthin-like pigment. At some stations 19'-hexanoyloxyfucoxanthin was more abundant
than fucoxanthin. 19'-hexanoyloxyfucocanthin has
recently been reported as present in the Southern
Ocean (Gieskes & Elbrachter 1986), in the tropical
Atlantic (Gieskes & Kraay 1986b) and dominant in the
North Sea during a bloom of the colonial prymnesiophyte Corymbellus aureus (Gieske & Kraay
1986a), during which it reached 7 times the concentration of fucoxanthin.
The origin of the fucoxanthin derivatives in Antarctic
waters is not yet clear, since very little is known of the
taxonomic distribution of these newly recognized pigments. Size fractionation of the Antarctic phytoplankton samples showed that both the 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin-like pigments were significantly concentrated in the < 5 pm
fraction, with most fucoxanthin being found in the
> 5 km fraction (Wright 1987), the latter probably
originating from diatoms (Stauber & Jeffrey unpubl.).
One source of the fucoxanthin derivatives is Likely to b e
the prymnesiophyte Phaeocystis pouchetii, which was
widespread in the study area during SIBEX 11 (Marchant & Wright unpubl.). Other sources of fucoxanthin
derivatives are also likely. A significant number of
nanoplanktonic flagellates (2 to 20 pm) were observed
in Antarctic water samples during this cruise (Marchant & Wright unpubl.), characterization of which has
not yet been completed. Booth & Marchant (1987) have
described 7 new species from Antarctic water phytoplankton within the newly described order Parmales in
the Chrysophyceae. Elucidation of the pigment composition of the Parmales and other unidentified forms
awaits successful isolation and culture. Pelagococcus
subviridis, another minute planktonic chrysophyte (2 to
3 pm), is widely distributed in the North Pacific Ocean
(Lewin et al. 1977),the East Australian Current (Vesk &
Jeffrey 1987) and Norwegian waters (Throndsen &
Kristiansen 1985) and can reach concentrations of 106
to 107 cells 1-l. Given the wide range of habitats in
which it has been found, it is quite possible that P.
subviridis and related organisms could be present in
Antarctic waters a s well, and contribute to the 19'butanoyloxyfucoxanthin-like distributions.
A few dinoflagellate genera are known to contain
combinations of fucoxanthin, 19'-butanoyloxyfucoxanthin and 19'-hexanoyloxyfucoxanth~n.However, dinoflagellates are unlikely to have been a major source of
fucoxanthin pigments in these Antarctic water samples. Although Gymnodinium spp. and other dinoflagellates were present in the study area, they were
never abundant (Marchant & Wright unpubl.).
265
Acknowledgements. We thank Mrs J . L. Stauber for isolating
the East Australian Current strains of Phaeocystis pouchem.
and Pelagococcus subvilidis, Mr P. Thomas and Dr S. I. Blackburn for the 2 Antarctic strains of Phaeocystis pouchetii, Dr
S I Blackburn for growing the cultures used in the present
work, Mr D. Le for the cell counts, Mr J. D Shearer for
assistance with the Southern Ocean field sampling, Mrs T.
Glaesner for HPLC assistance, and Dr N. Davies (University of
Tasmania) for the infrared spectrometry. We are indebted to
Dr G. M. Hallegraeff and Dr H. Marchant for helpful comments on the manuscricpt.
LITERATURE CITED
Arpin, N., Svec, W. A., haaen-Jensen, S. (1976). New fucoxanthin-related carotenoids from Coccolithus husleyi. Phytochemistry 15: 529-532
Berger, R., Liaaen-Jensen. S.. McAlister, V., Guillard, R. R. L.
(1977). Carotenoids of Prymnesiophyceae (Haptophyceae).
Biochem. Syst. Ecol. 5: 71-75
B j ~ r n l a n dT
, . , Pennington, F., Haxo, F. T., Liaaen-Jensen, S.
(1984). Carotenoids of Crysophyceae and Dinophyceae.
Abs. 7th Int IUPAC Symposium on Carotenoids, Munich,
p. 26
Bjernland, T., Tangen, K. (1979). Pigmentation and rnorphology of a marine Gyrodinium (Dinophyceae) with a major
carotenoid different from peridinin and fucoxanthln. J.
Phycol. 15- 457-463
Booth, B. C., Marchant, H. J. (1987). Parmales, a new order of
marine chrysophytes with descriptions of 3 new genera
and 7 new species. J. Phycol (in press)
Daley, R. J . , Gray, C. B. J . , Brown, S. R. (1973).A quantitabve,
semiroutine method for determining algal and sedimentary chlorophyll derivatives. J. Fish. Res. Bd Can. 30:
345-356
Foss, P,, Guillard, R. R. L., Liaaen-Jensen, S. (1984). Prasinoxanthin - a chemosystematic marker for algae. Phytochem.
23: 1629-1633
Gieskes, W. W. C . , Elbrachter, M. (1986).Abundance of nanoplankton-size chlorophyll-containing particles caused by
diatom disruption in surface waters of the Southern Ocean
(Antarctic Peninsula region). Neth. J. Sea Res. 20: 291-303
Gieskes, W. W. C.. Kraay, G. W (1983). Dominance of Cryptophyceae d u r ~ n gthe phytoplankton spnng bloom in the
central North Sea detected by HPLC analysis of pigments.
Mar. Biol. 75: 179-185
Gieskes, W. W. C., Kraay, G. W (1986a). Analysis of phytoplankton pigments by HPLC before, during and after mass
occurrence of the microflagellate Corymbellus aureus during the spring bloom in the open northern North Sea in
1983. Mar. Biol. 92: 45-52
Gieskes, W. W. C., Kraay, G. W. (198613). Floristic and physiological differences between the shallow and the deep
nanophytoplankton community in the euphotic zone of the
open tropical Atlantic revealed by HPLC analysis of pigments. Mar. Biol. 91: 567-576
Goodwin, T. W. (1955). Carotenoids. In: Peach, K., Tracey, M.
(ed.) Modern methods in plant analysis, Vol. 3. SpringerVerlag, Heidelberg, p. 272-31 1
Guillard, R. R. L., Murphy, L. S., Foss, P,, Liaaen-Jensen, S.
(1985). Synechococcus spp. as likely zeaxanthin-dominant
ultraphytoplankton in the North Atlantic. Lirnnol.
Oceanogr. 30: 4 1 2 4 1 4
Guillard, R. R. L., Ryther, J. H. (1962). Studies of marine
plankton diatoms. I. Cyclotella nana Hustedt and Detonula
confervacea (Cleve) Gran. Can. J. Microbial. 8: 229-239
266
Mar Ecol. Prog. Ser. 38: 259-266, 1987
Hallegraeff, G. M. (1981). Seasonal study of phytoplankton
pigments and species at a coastal station off Sydney:
importance of diatoms and the nanoplankton. Mar Biol.
61: 107-118
Hallegraeff, G . M., Jeffrey, S. W (1984).Tropical phytoplankton species and pigments of continental shelf waters of
North and North-West Australia. Mar Ecol. Prog. Ser. 20:
59-74
Hallegraeff, G . M . , Jeffrey, S W (1985) Description of new
chlorophyll a alteration products in marine phytoplankton.
Deep Sea Res. 32: 697-705
Jeffrey, S. W. (1974). Profiles of photosynthetic pigments in
the ocean using thin-layer chromatography. Mar. Biol. 26:
101-110
Jeffrey. S. W. (1976). A report of green algal pigments in the
Central North Pacific Ocean. Mar. Biol. 37: 33-37
Jeffrey, S. W. (1980). Cultivating unicellular marine plants.
CSIRO (Hobart, Tasmania) Fish and Oceanogr. A. Rep.
1977-1979, p. 22-43
Jeffrey, S. W. (1981).An improved thin-layer chromatographic
technique for marine phytoplankton pigments. hmnol.
Oceanogr. 26: 191-197
Jeffrey, S. W . , Hallegraeff, G . M. (1980) Studies of phytoplankton species and photosynthetic pigments in a warm
core eddy of the East Australian Current. I. Summer popul a t i o n ~ Mar.
.
Ecol. Prog. Ser. 3: 285-294
Jeffrey, S. W . , Hallegraeff, G M. (1987). Phytoplankton pigments, species and light climate in a complex warm-core
eddy of the East Australian Current. Deep Sea Res. 34: in
press
Jeffrey, S. W . , Humphrey, G . F. (1975). New spectrophotometric equations for determin~ngchlorophylls a, b, cl, and c2 in
higher plants, algae and natural phytoplankton. Biochem.
Physiol. Pflanz. 167: 191-194
Jeffrey, S. W., Vesk, M. (1981). The phytoplankton-systematics, morphology and ultrastructure. In: Clayton, M. N.,
ffing, R. J. (ed.) Marine botany - an Australasian perspective. Longman-Cheshire. Melbourne, p. 138-179
Jensen, A. (1966). Algal carotenoids. V Iso-fucoxanthinrearrangement product of fucoxanthin. Acta chem. scand.
29: 1728-1730
Ke, B., Imsgard, F., Kjosen, H., haaen-Jensen, S. (1970).
Electronic spectra of carotenoids at 77 K. Biochim. biophys.
Acta 210: 139-152
Lewin, J . , Norris, R. E.. Jeffrey, S. W., Pearson. B. E. (1977).An
aberrant chrysophycean alga Pelagococcus subviridis gen.
nov. et sp, nov. from the North Pacific Ocean. J . Phycol 13:
259-266
Loeblich. A. R. 111, Smith, V. E. (1968). Chloroplast pigments of
the marine dinoflagellate Gyrodinium resplendens. Lipids
3: 5-13
Mantoura, R. F. C . , Llewellyn, C. A. (1983). The rapid determinahon of algal chlorophyll and carotenold pigments and
their breakdown products in natural waters by reversephase high-performance liquid chromatography. Analytica chim. Acta 151: 297-314
Tangen, K., Bjarnland, T (1981) Observations on pigments
and morphology of Gyrodinium aureolum Hulbert, a
marine dinoflagellate containing 19'-hexanoyloxyfucoxanthin as the main carotenoid. J . Plankton Res. 3: 389-401
Throndsen, J., Kristiansen, S. (1985). Pelagococcus subvii-idis
Norris as a major component of the nanoplankton at Haltenbanken, Norwegian Sea in July 1982. Abst. Internat.
Phycological Congress, p. 160
Vesk, M., Jeffrey, S. W. (1987) Ultrastructure and pigments of
two strains of the picoplanktonic algae Pelagococcus subrids (Chrysophyceae). J. Phycol. 23: 322-336
Wright, S. W., Shearer, J. D. (1984). Rapid extraction and
high-performance liquid chromatography of chlorophylls
and carotenoids from marine phytoplankton. J .
Chromatog. 294: 281-295
Wright, S. W. (1987). The distribution and abundance of phytoplankton pigments during the SIBEX I1 cruise, Prydz
Bay, Antarctica, 1985. Australian National Antarctic
Research Expedition Research Note (in press)
This article was submitted to the editor; it was accepted for printing on April 15, 1987